The zonula occludens (ZO) or tight junction represents the transepithelial permeability barrier of the paracellular pathway in most vertebrate epithelia. This intercellular junction forms a gasket-like seal that encircles the cell at the intersection of the apical and lateral plasma membranes and thereby limits the diffusion of substances between lumenal and serosal components. The ZO is the apical most member of a series of intercellular junctions collectively known as the junctional complex. A preparation enriched for junctional complexes, including the structural elements of the ZO, has been generated from rodent liver. Antisera produced using this preparation as immunogen recognize the junctional complex region of hepatocytes as assayed by immunofluorescent staining. These antisera also stain the junctional region of other epithelial tissues including the human colonic cell line, HT-29. One particular monoclonal antibody has been shown to react on Western blots with a 260kd polypeptide (ZO-260). This antibody localizes at the ultrastructural level at the points of membrane contact at the ZO. It is proposed here to further localize this protein using immunonegative stain electron microscopy and to characterize its tissue and species distribution as well as its topographical orientation. Attempts will be made to purify in ZO-260 using immuno-affinity chromatography and to then reconstitute the ZO structure in artificial liposomes. The purified ZO-260 will also be used to generate additional immunological probes. These probes will be utilized in experiments aimed at a physiological and functional characterization of the ZO in situ. In addition, screening of mouse liver cDNA libraries for the ZO-260 gene is proposed. The original junctional complex enriched fraction will continue to be used as immunogen to obtain additional monoclonal antibodies directed toward other ZO- and junctional complex-associated proteins. Particular effort will be made to procure antibodies which recognize the junctional region of the HT-29 cell line because this system is amenable to analysis of junction assembly and function . It is hoped that in this manner a library of antisera can be produced, resulting in a structural and functional catalog of the molecular constituents of the junctional complex.